PCR problem

R. Jayakumar jakku at mrna.tn.nic.in
Wed Feb 7 07:24:16 EST 2001


I helped out  a similar problem with a friend of mine.   In that case, I
found that the forward primer was behaving as the reverse primer too due to
a near identical binding site downstream of the first site.  In the
subsequent PCR cycles, this becomes specific.  You should try sequencing the
0.5 kb fragment and analyse using GCG or something else.  You can also send
the sequence to me and I can help you out.
    bye and best of luck
jayakumar
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R. JAYAKUMAR
CSIR- Senior Research Fellow
Dept. of Molecular Microbiology,
School of Biotechnology,
Madurai Kamaraj University,
Madurai - 625 021.
India
email: jakku at linuxfan.com
tel: +91-452-858471-374
efax: (603)-688-4665
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"Life is a comedy for those who think and a tragedy for those who
feel" --Emerson
----- Original Message -----
From: ""divya venugopal"" <divyavenugopal at hotmail.com>
To: <methods at hgmp.mrc.ac.uk>
Sent: Tuesday, February 06, 2001 10:38 PM
Subject: PCR problem


> Can anybody help me out with this:
>
> I'm trying to amplify a 1.5kb region from the origin of replication of a
> plasmid from Mycobacterium avium-intracellulare.  The primers have been
> designed from the published sequence of the origin of replication.  The
> calculated annealing temperature of the primers is 67 degree C.  However i
> did not get any results at that temperature.  I got an amplicon at 55
degree
> C.  The size works out to be 0.5kb.  What could this be as i should be
> getting a 1.5 kb band. How is it possible to get the expected band.
>
> thank you,
>
> divya
>
>
> >From: Tim Spahlinger <txs at po.cwru.edu>
> >To: methods at hgmp.mrc.ac.uk
> >Subject: Re: primers problem
> >Date: Tue, 06 Feb 2001 10:42:48 -0500
> >
> >
> >
> >Tina wrote:
> >
> >
> >>Can we dilute primers in some buffer instead of water. If yes, which
> >>buffer
> >>?
> >>Is there a special task with porcine primers?
> >>Does water have any effect on the primers?
> >>Any suggestions ?
> >>
> >>Thanks a lot !
> >
> >I've used TE buffer to reconstitute and freeze (-20 C)
> >commercially prepared primers.
> >
> >
> >
>
> _________________________________________________________________________
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>
>
> ---
>
>
>


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